Mutations in Ebf1, Pax5 and Ikaros, three transcription factors that form an interrelated gene network required to initiate B cell development, are frequently mutated in progenitor B-ALL. Likewise, the transcription factor STAT5 is frequently activated in B-ALL. Importantly, mutations in the Ebf1/Pax5/Ikaros network, most notably for Ikaros, have been shown to correlate with poor outcome in human ALL; we and others have observed a similar correlation between STAT5 activation in ALL and poor prognosis. However, whether mutations in the Ebf1Pax5/Ikaros network, or STAT5 activation, drive transformation or are merely passengers in this process remains unclear. We recently obtained an important insight to this question by crossing mice expressing a constitutively active form of STAT5b (called Stat5b-CA) to mice heterozygous for Ebf1 or Pax5. Stat5b-CA mice develop ALL with very low penetrance (1-2%) while Pax5 and Ebf1 mice show no predisposition to develop ALL. In contrast, 100% of Stat5b-CA x Pax5 and Stat5b-CA x Ebf1 mice developed ALL with an average age of onset of 55 and 108 days after birth, respectively. Importantly, Stat5b-CA x Ebf1 and Stat5b-CA x Pax5 leukemias did not lose the other allele of either Ebf1 or Pax5, respectively. Our hypothesis is that genes that make up the Ebf1/Pax5/Ikaros network do not act as classical tumor suppressor genes but rather function as part of an integrated tumor suppressor gene network. In this model loss of any two alleles of any of the genes in the network, as opposed to both alleles of the same gene is sufficient for loss of tumor suppressor function. We further propose that STAT5 activation is needed to pair with loss-of-function mutations in the Ebf1/Pax5/Ikaros network to initiate transformation. In this grant application, we will test these hypotheses using a Sleeping beauty transposon-based mutagenesis strategy. In addition, we will test whether defects in the Ebf1/Pax5/Ikaros network in human ALL correlate with increased STAT5 signaling. Finally, we will also use the Sleeping Beauty genetic screen to identify other genes that cooperate with defects in the Ebf1/Pax5/Ikaros network and STAT5 activation to initiate and promote progenitor B-ALL. These studies should provide important insights into the role that STAT5 and the Ebf1/Pax5/Ikaros network play in causing B-ALL as well as identify additional genes that play key roles in the process of transformation.